Methods for measuring and optimizing the structure, location, and activity of natural and engineered microcomparments, organelles, and macromolecules

Abstract

A novel method of diluting the structures in the cell population, such that individual cells, dependent on the activity of the structures, become single measurement devices. This can be applied to all Bacterial Microcomparments (“BMCs”), organelles, and macromolecules, and could provide a universal method for the design of novel ones and understanding of the diverse structures. In one aspect the present invention provides A method of creating a bacterial strain with inducible and detectable carboxysomes. The method includes the steps of incorporating a labeled carbon-fixation enzyme into the genome of a bacterium; deleting all or a portion of the ccm operon from the bacterium; and reintroducing a ccm operon comprising an inducible promoter to create a Δccm+ strain.

What is claimed is: 
     
       1. A method of controlling growth, expression, or carboxysome number in a bacterial strain comprising the steps of:
 providing a bacterial strain having a ccm operon under the control of an inducible promoter; 
 contacting the bacterial strain with an agent that induces the promoter, whereby inducing the promoter increases growth, expression, or carboxysome number; 
 removing the agent from the bacterial strain; and 
 maintaining the bacterial strain in ambient CO 2  or other CO 2  concentration wherein carboxysome expression is inhibited, whereby continued growth and division of the bacterial strain results in a decrease in the number of carboxysomes in progeny cells. 
 
     
     
       2. The method of controlling growth, expression, or carboxysome number in a bacterial strain according to  claim 1  wherein all or a portion of the native ccm operon has been deleted. 
     
     
       3. The method of controlling growth, expression, or carboxysome number in a bacterial strain according to  claim 1  further comprising the step of maintaining the bacterial strain in ambient CO 2  or other CO 2  concentration wherein carboxysome expression is inhibited, prior to the step of contacting the bacterial strain with the agent that induces the promoter. 
     
     
       4. A method of controlling carboxysome number, growth, or expression in a bacterial strain comprising the steps of:
 providing a bacterial strain having a labeled carbon-fixation enzyme and genetically-engineered to have all or a portion of a ccm operon of the bacterial strain under the control of an inducible promoter; 
 maintaining a population of the bacterial strain in ambient CO 2  or other concentration of CO 2  wherein production of carboxysomes in the population is inhibited and expression from the ccm operon in the bacterial strain is not induced; 
 contacting the population of the bacterial strain with an agent that induces the inducible promoter of the ccm operon, wherein inducing the promoter increases expression from the ccm operon whereby increasing expression from the ccm operon increases carboxysome numbers in the population; 
 removing the agent from the population of the bacterial strain; 
 maintaining the population of the bacterial strain in ambient CO 2  or other CO 2  concentration wherein expression from the ccm operon is inhibited, whereby continued growth and division of the population of bacterial strain results in a decrease on the number of carboxysomes in progeny cells in the population; 
 detecting puncta in the population of cells resulting from labeled enzymes within in the carboxysomes of the bacterial strain, whereby punctum correspond to single carboxysomes; 
 identifying a bacterial cell within the population containing only one punctum per cell; and 
 following the identified bacterial cell through a plurality of timepoints. 
 
     
     
       5. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 4  further comprising the step of measuring the growth of the cell at two or more timepoints, wherein the growth is an indicator of carboxysome activity. 
     
     
       6. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 4  wherein all or a portion of the native ccm operon has been deleted. 
     
     
       7. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 4  further comprising the step of measuring one or more parameters of the identified bacterial cells at a plurality of timepoints. 
     
     
       8. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 7  wherein one of the measured parameters is cell length. 
     
     
       9. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 8  further comprising the step of contacting the cell with one or more agents and comparing the measured parameter to the parameter in an uncontacted control. 
     
     
       10. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 4  wherein puncta are detected by visualization. 
     
     
       11. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 10  wherein visualization is facilitated by fluorescence of the label. 
     
     
       12. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 4  wherein following the identified bacterial cell is performed by time-lapse fluorescence microscopy to facilitate tracking of fluorescently-labeled carboxysomes. 
     
     
       13. The method of controlling carboxysome number, growth, or expression in a bacterial strain according to  claim 4  wherein the carbon-fixation enzyme is 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO). 
     
     
       14. A method of controlling bacterial growth, expression, or microcompartment number in a bacterial strain comprising the steps of:
 providing a population of the bacterial strain genetically-engineered to have all or a portion of a ccm operon of the bacterial strain under the control of an inducible promoter; 
 contacting the population of the bacterial strain with an agent that induces the inducible promoter of the ccm operon, wherein inducing the promoter increases expression from the ccm operon whereby increasing expression from the ccm operon increases carboxysome numbers in the population; 
 removing the agent from the population of the bacterial strain; 
 maintaining the population of the bacterial strain in ambient CO 2  or other CO 2  concentration wherein expression from the ccm operon is inhibited, whereby continued growth and division of the population of bacterial strain results in a decrease on the number of carboxysomes in progeny cells in the population; and 
 following the identified bacterial cell through a plurality of timepoints.